Chronic alcohol abuse produces lasting changes in brain function that are manifested as tolerance, physical dependence, craving, and other behavioral changes. The Mayfield Project will test the overall hypothesis that these changes are due to the co-ordinated regulation of alcohol-responsive genes by small non-coding RNAs. This Project will explore the role of these regulatory RNAs using several innovative approaches to alcohol research, including profiling of all known miRNAs, next generation sequencing of unique small RNAs, prediction and validation of microRNAs (miRNAs):mRNA target interactions, expression patterns of miRNAs which may act in combination to regulate mRNA expression and delivery of selected alcohol-related miRNAs to brain. Three Specific Aims are proposed: Aim 1 will test the hypothesis that miRNA expression profiling and next generation sequencing will identify unique alcohol-sensitive small RNAs in the nucleus accumbens and ventral tegmental area of mouse brain. Subsequent RT-PCR analysis will validate and define which of these are involved in mediating the effects of alcohol consumption in mouse models. Aim 2 will test the hypothesis that differentially expressed miRNAs are co-ordinately expressed and act in combination by direct interaction with their predicted targets to regulate gene expression. The changes in expression that occur as a result of miRNA regulation are functionally important and may underlie alcohol's actions in the brain. Aim 3 will test the hypothesis that over-expression of select miRNAs in mouse brain, either individually or in combination, will alter drinking phenotypes, and these changes will be correlated with specific patterns of gene expression in the nucleus accumbens and ventral tegmental area of mouse brain.